Integrating Pt nanoparticles with 3D Cu2- x Se/GO nanostructure to achieve nir-enhanced peroxidizing Nano-enzymes for dynamic monitoring the level of H2O2 during the inflammation

The treatment of wound inflammation is intricately linked to the concentration of reactive oxygen species (ROS) in the wound microenvironment. Among these ROS, H2O2 serves as a critical signaling molecule and second messenger, necessitating the urgent need for its rapid real-time quantitative detection, as well as effective clearance, in the pursuit of effective wound inflammation treatment. Here, we exploited a sophisticated 3D Cu2- x Se/GO nanostructure-based nanonzymatic H2O2 electrochemical sensor, which is further decorated with evenly distributed Pt nanoparticles (Pt NPs) through electrodeposition. The obtained Cu2- x Se/GO@Pt/SPCE sensing electrode possesses a remarkable increase in specific surface derived from the three-dimensional surface constructed by GO nanosheets. Moreover, the localized surface plasma effect of the Cu2- x Se nanospheres enhances the separation of photogenerated electron-hole pairs between the interface of the Cu2- x Se NPs and the Pt NPs. This innovation enables near-infrared light-enhanced catalysis, significantly reducing the detection limit of the Cu2- x Se/GO@Pt/SPCE sensing electrode for H2O2 (from 1.45 μM to 0.53μM) under NIR light. Furthermore, this biosensor electrode enables in-situ real-time monitoring of H2O2 released by cells. The NIR-enhanced Cu2- x Se/GO@Pt/SPCE sensing electrode provide a simple-yet-effective method to achieve a detection of ROS (H2O2、-OH) with high sensitivity and efficiency. This innovation promises to revolutionize the field of wound inflammation treatment by providing clinicians with a powerful tool for accurate and rapid assessment of ROS levels, ultimately leading to improved patient outcomes.


Introduction
Reactive oxygen species (ROS) play a vital role in wound inflammation treatment (1).Their reactive and destructive properties enable neutrophils and macrophages to phagocytose, thus aiding in inflammation management (2)(3)(4).Hydrogen peroxide (H 2 O 2 ) is a major secondary messenger in the treatment of inflammation and a typical ROS molecule in biomedical diagnostics (5,6).Over the past few years, the preponderance of evidence has established that H 2 O 2 has a particularly significant impact on the stress/inflammatory response and subsequent tissue/neuronal repair processes (7)(8)(9).The inherent characteristics of H 2 O 2 such as its ease of degradation, long half-life, universal present in all cells, and membrane/tissue permeability -dictate its functional role.Within a certain concentration range, H 2 O 2 performs multiple functions: (1) it stimulates proliferation of human fibroblasts and vascular endothelial cells; (2) it boosts macrophage inflammatory protein (MIP)-1a production; and (3) it promotes angiogenesis via vascular endothelial growth factor (VEGF) signaling and catalytic effects on keratin-forming cells (2,10).However, at elevated concentrations, H 2 O 2 can lead to cell necrosis by triggering pro-apoptotic proteins and ultimately causing cell death.It is evident that the physiological effects of H 2 O 2 are usually concentration-dependent; hence, there is a pressing need to explore effective methods for accurately detecting H 2 O 2 concentration in living cells (11).S e n s i n g t e c h n o l o g i e s s u c h a s fl u o r e s c e n ce ( 1 2) , chemiluminescence (13), colorimetry (14), photoelectrochemical sensing (15) and electrochemical sensing (16) have been developed for the detection of H 2 O 2 .Recently, electrochemical sensing technology, which offers the advantages of simple operation, fast response, high sensitivity, and high selectivity has been receiving immense research attention for real-time H 2 O 2 detection.To address the limitations of expensive and environmentally unstable catalase to the widespread application of sensors, there has been a growing interest in the study of biomimetic electrochemical H 2 O 2 sensors that possess peroxidaselike activity without the need for enzymes (17,18).
The advancement of efficient nano-enzymatic materials is paramount for expeditious in situ detection of H 2 O 2 (19).These materials, which range from noble metal nanoparticles like Au (20), Pt (21), Ag (22), etc., to metal oxides or compounds nanostructures such as CeO 2 (23), TiO 2 (24), MnO 2 (25), etc., exhibit exceptional hydrogen peroxidase activity.This activity not only effectively mitigates excess H 2 O 2 in the physiological environments, but also signifies their vast potential in applications like wound healing and inflammation therapy.Specifically, Pt NPs demonstrate promising applications in H 2 O 2 electrochemical sensors due to their excellent peroxidase-like activity (26,27).However, ensuring the uniform anchoring of these metal nanoparticles on smooth electrodes remains a significant challenge for electrochemical detection of H 2 O 2 .Fortunately, numerous studies have established that carbonbased materials can be used as support structures for growing and anchoring metal particles, thereby preventing their aggregation (28).Carbon nanosubstrates such as carbon nanotubes (29), graphene (30) and carbon fibers (31) can even facilitate the spontaneous generation of metal nanoparticles on the substrate, eliminating the need for additional reducing agents.As an example, Tong et al (32).reported a self-terminating chemical deposition method to prepare surfactant-free monodisperse Pt nanoparticle (NP)-modified carbon fiber microelectrodes (Pt-NP/CFE) for electrochemical detection of hydrogen peroxide (H 2 O 2 ) released by living cells.This method represents a significant breakthrough in the field of nano-enzymatic materials development, paving the way for more effective and precise in situ detection of H 2 O 2 .However, the enhancement of stability and catalytic activity of metal nanoparticles remains a formidable challenge for their implementation in the expeditious and precise detection of trace amounts of H 2 O 2 .To achieve rapid detection of minute amounts of H 2 O 2 , extensive research has been conducted on the development of composite nanoenzymes.Innovative researchers have devised various copper-based nanomaterials, including CuO (33), Cu-MOF (34), and CuS (35), and evaluated their efficacy in enzyme-like catalysis.Copper-based nanomaterials hold high research value for their application in H 2 O 2 detection.Furthermore, copper-based nanomaterials exhibit unique optical properties.In particular, copper selenide nanocrystals (Cu 2-x Se), as a p-type semiconductor nanomaterial, generate a profusion of photogenerated electron-hole pairs under irradiation of near-infrared light by localized surface plasmon resonance (LSPR) in response to oscillating electromagnetic field interactions of light (36).Moreover, the semiconductor-metal heterojunction formed by the union of copper selenide and metal nanoparticles abbreviates the band gap of the material, thereby augmenting its catalytic activity.
Herein, we have developed sensing electrodes, made from Cu 2-x Se/GO@Pt composites, for rapid and precise in situ detection of H 2 O 2 (Scheme 1).Initially, we employed our previously established method (37) to generate Cu 2-x Se nanospheres containing copper defects.Subsequently, we integrated GO nanosheets as a substrate, ensuring the nanospheres were firmly anchored to the electrodes and facilitating the electrodeposition of Pt nanoparticles.Meanwhile, the three-dimensional structure of the GO nanosheets on the electrode surface not only provides a larger specific surface area, but also offers more efficient electron and ion transport paths, ultimately enhancing the catalytic performance of the sensing electrode.In addition, the unique photoelectric effect of Cu  China).Phosphate buffered saline (PBS) was purchased from BioInd (Gibco).All chemicals and reagents were used as received without any further purification.All solutions were prepared using ultrapure water (18.2MW).Graphene oxide (GO) powder and Mxene powder were both obtained from Sigma-Aldrich (St. Louis, USA).

Synthesis of Cu 2-x Se NPs
Cu 2-x Se NPs were prepared by the previously reported situ reduction method.Briefly, 1.6 mL PSS (10 mg mL -1 ) and 5.5 mL DI water were added into a round-bottom flask which stirred in a 30°C water bath, then 0.3 mL Vc (0.4 M) and 0.1 mL SeO 2 (0.2 M) were added in sequence, stirring at 30°C for 20 min.A mixed solution of 0.1 mL CuSO 4 •5H 2 O (0.4 M) and 0.4 mL Vc (0.4 M) were added in the next step.Then, the mixed solution was strongly stirred at 45°C for 8h to obtain a dark green dispersion, centrifuged at 12000 rpm for 5 min, and suspended in DI water to obtain Cu 2-x Se solution.Next, the Cu 2-x Se solution freeze dried in vacuum environment, and it was redispersed in DI water (1 mg mL -1 ) at 4°C for later use.

Fabrication of the Cu 2-x Se/GO@Pt/SPCE Sensing Electrodes
Firstly, several screen-printed carbon electrodes (SPCE) were prepared, washed with DI water and then dried for use.In a general way, 10 mL Cu 2-x Se NPs (1 mg mL -1 ), 2.5 mL GO (1 mg mL -1 ), and 7.5 mL DI water were uniformly mixed together, and then 10 mL of the mixture (Cu 2-x Se/GO) was dropped on a bare SPCE, followed by drying at 37°C.After that, Pt NPs were electrodeposited on Cu 2-x Se/ GO/SPCE in 5 mL mixture of 1 mM H 2 PtCl 6 solution to obtain Cu 2-x Se/GO@Pt/SPCE.For comparison, the same method was used to prepare Cu 2-x Se/Mxene@Pt/SPCE, Cu 2-x Se @Pt/SPCE, and prepared Cu 2-x Se/SPCE without electrodeposition.

Characterization of Cu 2-x Se/GO@Pt/SPCE sensing electrodes
Scanning electron microscopy (SEM) was used to analyze the surface morphology of Cu 2-x Se/GO@Pt/SPCE and the surface distribution of Pt nanoparticles.X-ray photoelectron spectroscopy (XPS) was used to analyze the chemical states of elements on the surface of the prepared sensor electrodes.

Electrochemical measurements
All the electrochemical tests were performed using the CHI760E electrochemical workstation.The electrodeposition process is carried out for 200 s at a potential of −0.2 V using chronocurrent method (i-t).In order to characterize the electrochemical behavior of Cu 2-x Se/GO@Pt/SPCE, CV and EIS were recorded in a 5 mM [Fe (CN) 6 ] 3 − /4− solution containing 0.1 M KCl.When the electrocatalytic performance of H 2 O 2 was tested by Cu 2-x Se/GO@ Pt/SPCE, CV and i-t were monitored at 0.1 M PBS (pH = 7.4).The test parameters of CV: the operating voltage range is -0.8~0.8V, and the scanning rate is 20-300 mV s -1 .The linear response of the detection electrode to H 2 O 2 in the i-t test was performed at an optimal operating voltage of -0.4 V.

Electrochemical detection of H 2 O 2 released from cells
The L929 cells (Shanghai Yihe Biotechnology Co., Ltd) were cultured in 5% CO 2 in 75 cm 2 culture flask containing MEM medium, 1% penicillin and streptomycin and 10% (v/v) horse serum at 37 °C.Unlike L929 cells, RAW264.7 (Shanghai Yihe Biotechnology Co., Ltd) cells need to be cultured in 75 cm 2 culture flasks containing DMEM medium, 10% FBS and 1% double antibody at 37 °C and 5% CO2Wash 2 times in PBS (0.01 M, pH=7.4),digest the cells, and centrifuge at 1000 rpm for 3min.Resuspend the cells and counted the cells by calculating instrument.When the current keeps stable, gently stir the cell suspension.Then, the N-Formyl-Met-Leu-Phe (fMLP, Sigma) was added to the cell suspension with different concentration, which can motivate cells generation of H 2 O 2 and have no interference to the detection of H 2 O 2 .The amperometric current response of H 2 O 2 in about 7*10 7 L929 cells or 8*10 7 RAW 264.7 cells in 10 mL of PBS with the bare SPCE and Cu 2-x Se/GO@Pt/SPCE, and the electrode was recorded at -0.4 V.

Results and discussion
Characterization of the As-prepared Cu 2-x Se/GO@Pt/SPCE sensing electrode The morphology and structure of the materials and electrodes were characterized by transmission electron microscopy (TEM) and scanning electron microscopy (SEM).As shown in Supplementary Figure S1, TEM images of Cu 2-x Se nanospheres proved that they had a uniform spherical morphology with a diameter of about 40 nm and a smooth surface.The SEM images of the bare screen-printed carbon electrodes (SPCE) electrode exhibited that the electrode possesses a rough and porous micro-morphology, which is favorable for modifying the nano-electrode materials (Supplementary Figures S2A, B).However, as shown in Supplementary Figure S2C, when only Cu 2-x Se NPs were modified on the SPCE, the electrode surface was not completely covered by the nanospheres.In contrast, when the Cu 2-x Se/GO mixture was added dropwise to the electrode, the Cu 2-x Se NPs were evenly distributed across the graphene oxide nanosheets, and the complex completely covered the SPCE surface (Supplementary Figure S2E).The incorporation of GO nanosheets provided a reliable three-dimensional (3D) framework for Cu 2-x Se NPs and contributed to their anchoring to the electrodes.SEM images of Cu 2-x Se/GO@Pt/SPCE obtained after modifying Pt nanoparticles (Pt NPs) on Cu 2-x Se/GO/SPCE by electrodeposition were depicted in Figures 1A-C.The image results showed that the electrode modified with Pt NPs still presented a 3D porous shape, indicating that the modification of Pt NPs didn't affect the morphology and structure of GO and Cu 2-x Se NPs.The 3D porous structure formed by GO nanosheets and Cu 2-x Se NPs as a substrate for the electrodeposition of Pt NPs reduced the aggregation of nanoparticles, promoted a robust anchoring of nanoparticles, and provided a reducing agent for the reduction of monomeric Pt.Furthermore, the EDS elemental mapping was carried out to observe the elemental compositions of Cu 2-x Se/GO@Pt/SPCE electrode, in which the Pt signal confirmed the successful loading of the Pt NPs in the Cu 2-x Se/GO structure (Figure 1D).
In order to study the elemental chemical composition and valence states of Cu 2-x Se/GO@Pt/SPCE, we characterized the Frontiers in Immunology frontiersin.orgelectrode using by X-ray photoelectron spectroscopy (XPS).The XPS spectra revealed the presence of C, Cu, Se, and Pt elements in the Cu 2-x Se/GO@Pt/SPCE (Supplementary Figure S3A).The spectra of the Cu 2p orbital indicated the coexistence of both Cu 2 + and Cu + in Cu 2-x Se NPs (Figure 2A) and Cu 2-x Se/GO@Pt/SPCE (Figure 2B).Meanwhile, the proportion of Cu 2+ increased from 23.88% to 31.73% after the deposition of Pt NPs, consistent with the Cu + oxidation process.Compared to Cu 2-x Se NPs, there were obvious negative shifts for the Cu 2+ and Cu + binding energies of Cu 2-x Se/GO@Pt/SPCE, indicating a strong interaction between Pt NPs and Cu 2-x Se NPs.The high-resolution XPS spectrum of Cu 2- x Se/GO@Pt/SPCE showed peaks of C1s at 284.05, 284.50, and 285.0 eV corresponding to C=C/C-C, C-O, and C=O bonds (Figure 2C) (38).Moreover, the Se 3d XPS spectrum of the Cu 2-x Se NPs and Cu 2-x Se/GO@Pt/SPCE exhibited two main peaks (Se 3d 5/2 and Se 3d 3/2 ), indicating that that the valence state of Se element was not affected by the electrodeposition process of Pt NPs (Supplementary Figures S3B, C).Additionally, Pt 0 peaks were observed at the binding energies of 74.4 eV and 71.01 eV, with a Pt 0 ratio of 72.95% (Figure 2D), confirming the successful deposition of Pt NPs.Based on the above analysis, we have successfully prepared a Cu 2- x Se/GO@Pt/SPCE sensing electrode with a unique 3D porous structure, providing a faster conductive network for the electrochemical detection of H 2 O 2 .This advancement paves the way for future applications in sensing technologies.

Electrochemical characterization of Cu 2-x Se/GO@Pt/SPCE sensing electrode
To evaluate the electrochemical performance of SPCE, Cu 2-x Se/ SPCE, Cu 2-x Se@Pt/SPCE, Cu 2-x Se/Mxene@Pt/SPCE and Cu 2-x Se/ GO@Pt/SPCE, typical cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) has been applied to thoroughly characterize the electrodes (Figure 3A; Supplementary Figure S4).As shown in Figure 3A, a pair of Fe 2+ / 3+ redox peaks could be observed on the CV curves of all electrodes in the range of 0-0.2 V.However, the redox peaks of all electrodes except the bare SPCE exhibited wider peak potential differences and lower peak currents.Apparently, these electrodes also exhibited a pair of Cu 2+ /Cu + redox peaks in the range of 0.4-0.6V, which influenced the peak width and peak current of Fe 2+ / 3+ redox peak.Among them, Cu 2-x Se/ GO@Pt/SPCE exhibited the highest peak current in the range of 0.4-0.6V, highlighting its excellent electrochemical performance.Additionally, Supplementary Figure S4 displays the EIS curves of all the aforementioned electrodes.The EIS curves of the Cu 2-x Se/GO@ Pt/SPCE in the line part representing Warburg impedance (Rw) exhibited the steepest slope, indicating that electrolyte ions had the smallest diffusion resistance in the pores of the electrode, thereby facilitating electrochemical reactions.These findings highlight the significance of material design and engineering in enhancing electrochemical performance.Furthermore, the electrocatalysis of H 2 O 2 by the prepared electrodes mentioned above was investigated in 10 mL 0.01 M PBS (pH 7.4) system using both CV and i-t methods.The CV curves of each electrode in PBS buffer containing 5 mM H 2 O 2 , as displayed in Figure 3B, clearly indicate that the Cu 2-x Se/SPCE without electrodeposition of Pt NPs exhibited no distinct characteristic peak of current response.In contrast, the other three electrodes showed well-defined reduction peaks at -0.32 V (Cu 2-x Se@Pt/SPCE), -0.3 V (Cu 2-x Se/Mxene@Pt/SPCE), and -0.4 V (Cu 2-x Se/GO@Pt/SPCE).These findings convincingly demonstrate that Pt NPs possess a profound catalytic effect on H 2 O 2 .Notably, the Cu 2-x Se/GO@Pt/ SPCE exhibited the largest reduction peak current.Subsequently, i-t tests were conducted on the corresponding sensing electrodes at the reduction potentials observed in the CV curves.The i-t curves presented in Figure 3C align with the CV curves, offering consistent results.With the continuous addition of 0.1 mM H 2 O 2 , the current signal presented a stepped response, and the Cu 2-x Se/ GO@Pt/SPCE displayed the highest response current.Obviously, the above results confirmed that the catalytic reduction of H 2 O 2 by the sensing electrode was mainly attributed to the Pt NPs on the electrodes.When comparing the two substrate materials, Mxene and GO nanosheets, it is evident that the latter is a more suitable support material for Cu 2-x Se NPs and an effective electrodeposition reducer for Pt NPs, thanks to its constructed three-dimensional porous structure.Furthermore, except for Cu 2-x Se/SPCE, the reduction peak current signals of CV curves of other electrodes gradually increased with increasing H 2 O 2 concentration (0-5 mM), while the reduction peak potential shifted slightly in the negative direction (Figure 3D; Supplementary Figure S5).
In order to gain a deeper understanding of the catalytic kinetic process of H 2 O 2 on the electrode, we conducted CV studies to measure the catalytic current signals of 5 mM H 2 O 2 on Cu 2-x Se/ GO@Pt/SPCE at different scanning rates.As displayed in Figure 3E, a gradually increasing scan rate ranging from 20 to 300 mV s -1 resulted in an increase in the reduction current.Additionally, a strong linear relationship was observed between the peak current and the square root of the scanning rate (see Figure 3F), indicating that the catalytic process is highly dependent on the scan rate.The equation describing this relationship is provided below: i(mA) = 283:60v 1 2 (V=s 1=2 ) + 13:67 (R 2 = 0:993) By scrutinizing the catalytic reduction reaction of H 2 O 2 on Cu 2-x Se/GO@Pt/SPCE, we've ascertained that it's an irreversible process, controlled by diffusion.This observation underscores the exceptional electrocatalytic activity of Cu 2-x Se/GO@Pt/SPCE.Consequently, we selected this material for a more detailed examination of its sensitivity in detecting H 2 O 2 .
To optimize detection parameters, we conducted chronoamperometry (i-t) to study the effect of Cu 2-x Se/GO@Pt/ SPCE on the catalytic performance of H 2 O 2 at different operating potentials (-0.36 V~-0.4 V).As depicted in Figure S6A, as the voltage increased from -0.36 V to -0.4 V, the response current of Cu 2-x Se/GO@Pt/SPCE to H 2 O 2 rises gradually.However, upon further voltage increment, the response current does not continue to climb, and even slightly decreased.Clearly, this suggests that Cu 2-x Se/GO@Pt/SPCE reaches peak catalytic performance for H 2 O 2 at -0.4V.Therefore, -0.4V was chosen as the operating potential for the subsequent experiments.Additionally, we evaluated the linear relationship between Cu 2-x Se/GO@Pt/SPCE's response to H 2 O 2 at an operating potential of -0.4V through i-t measurements.At the same time, considering the optical characteristics of Cu 2-x Se NPs, the method of near infrared illumination was used to improve the catalytic effect of Cu 2-x Se/GO@ Pt/SPCE on H 2 O 2 .In addition, in order to investigate the effect of the content of Pt NPs on the electrochemical performance of the sensor electrode enhanced by NIR, we tested the i-t curves of the sensor electrode with different deposition times.Supplementary Figure S7A displays the amperometric response of Cu 2-x Se/GO@Pt/SPCE with successive injection of 100 mM H 2 O 2 at different deposition times with 808 nm laser.When the deposition time is greater than 200s, the growth of current signal tends to be stable.Thus, 200 s of deposition time was used for subsequent experiments.As Figure 4A clearly illustrates, regardless of whether 808 nm laser irradiation was used, the continuous addition of H 2 O 2 solution with increasing concentration to a 10 mL 0.01M PBS solution led to a rapid steptype response of the reduction current of Cu 2-x Se/GO@Pt/SPCE.This response reached a stable state within a few seconds (<5s), highlighting the electrode's exceptional response speed and its ability to catalyze H 2 O 2 rapidly.Obviously, under 808 nm laser irradiation, the Cu 2-x Se/GO@Pt/SPCE displayed a significantly larger response current to H 2 O 2 , indicating that the laser could enhance the electrode's catalytic effect on H 2 O 2 .Moreover, Figure 4B presents the i-t curves of the low concentration region (10~50 mm) in Figure 4A, in which the improvement of H 2 O 2 catalytic performance of Cu 2-x Se/ GO@Pt/SPCE by 808 nm laser at low concentration could be more clearly seen.Figure 4C further illustrates the linear relationship curves between the current signal and H 2 O 2 concentration, both without and with 808 nm laser irradiation.The corresponding linear fitting equation is as follows: i(mA) = 0:033C H 2 O 2 (mM) − 0:033(R 2 = 0:990) without laser) and i(mA) = 0:045C H 2 O 2 (mM) − 0:311(R 2 = 0:997), with 808 nm laser).Based on the above linear fitting equation, the detection limits without laser irradiation and under 808 nm laser irradiation could be calculated as 1.45mM and 0.53 mM, respectively (LOD = 3s

S d
).Compared to the other electrochemical sensors and other detection methods in Supplementary Table S1, a relatively low detection limit is obtained at Cu 2-x Se/GO@Pt/SPCE.findings underscore the critical role of near-infrared illumination in enhancing the H 2 O 2 catalytic performance of Cu 2-x Se/GO@Pt/ SPCE.This exceptional enhancement effect under NIR light is primarily attributed to the local surface plasmon resonance (LSPR) of Cu 2-x Se NPs. which generated more charge carriers on the electrode surface under laser irradiation, and these charge carriers then reacted with H 2 O 2 to produce a larger response current.
In practical applications, the selectivity and reproducibility of the sensor are paramount considerations.We evaluated these factors by monitoring the interference of several potential interfering substances (glycine (Gly), citric acid (CA), ascorbic acid (AA), urea, glutathione (GSH)) on the detection of H 2 O 2 using Cu 2-x Se/GO@Pt/SPCE with i-t curve.As illustrated in Figure 4D, even at higher concentrations (1 mM), Gly, CA, AA, urea and GSH did not produce any discernible amperometric response when compared to H 2 O 2 (100 mM).These results suggested the remarkable selectivity for H 2 O 2 electrochemical detection.Furthermore, we measured the current response of five Cu 2-x Se/GO@Pt/SPCE sensing electrodes to 300 mM H 2 O 2 with i-t curves and repeated the test six times for each electrode (Supplementary Figure S8).The relative standard deviation (RSD) among electrodes was calculated to be 8.27%, indicating excellent reproducibility of the sensor.
Detection of H 2 O 2 released from living cells at Cu 2-x Se/GO@Pt/SPCE The concentration of H 2 O 2 in living cells is a significant biological parameter associated with wound inflammation treatment.Therefore, in this study, L929 cells and RAW 264.7 cells were selected as model cells, and the fLMP drug was used to stimulate the release of H 2 O 2 within them.The microscopic images of L929 cells and RAW 264.7 cells displayed in Supplementary Figure S9, L929 cells exhibit fibroblast-like characteristics and grow close to the cell wall, while RAW 264.7 cells possess a small, translucent, and rounded morphology.Figure 5A provides a schematic diagram demonstrating how fLMP stimulates cells to produce H 2 O 2 , which is then rapidly detected followed by rapid detection of H 2 O 2 by Cu 2-x Se/GO@Pt/SPCE.By utilizing Cu 2-x Se/ GO@Pt/SPCE as the sensing electrode, the cells were stimulated to release H 2 O 2 upon the addition of the fLMP drug to two distinct cell suspensions, and the signal of H 2 O 2 was then determined by the i-t measurement (Figures 5B, C).When compared to a bare SPCE electrode, the Cu 2-x Se/GO@Pt/SPCE sensing electrode exhibited significant reduction currents for H 2 O 2 produced under different concentrations of fLMP (20 mM, 40 mM) stimulation within both cell suspensions, suggesting that the Cu 2-x Se/GO@Pt nano-mimetic enzyme exhibits remarkable catalytic activity towards H 2 O 2 .The reduction currents generated by the bare SPCE electrode and the Cu 2-x Se/GO@Pt/SPCE sensing electrode stimulated with the same concentration of fLMP (40 mM) in both cell suspensions are shown in Supplementary Figure S10, and there is a strong significant difference between the two sets of data, which further suggests that the catalytic reduction of H 2 O 2 is mainly attributed to the Cu 2- x Se/GO@Pt nanomimetic enzyme.In particular, the i-t curve of the Cu 2-x Se/GO@Pt/SPCE retained a significant H 2 O 2 response peak even when a lower concentration (8 mM) of fLMP solution was added to the L929 cell suspension (Supplementary Figure S11).This further highlights the outstanding catalytic activity of the Cu 2-x Se/ GO@Pt nanomimetic enzyme.The magnitude of the current response across the curves demonstrated that the amount of H 2 O 2 produced by the cells increased in direct correlation with the concentration of fLMP.Additionally, as shown in Figure 5D, the magnitudes of the reaction currents in both cell suspensions were 0.046 mA, 0.066 mA (L929) and 0.029 mA, 0.123 mA (RAW 264.7) when fLMP was added in the amounts of 20 mM and 40 mM, respectively, indicating that fLMP promotes the dose-dependent release of H 2 O 2 from the cells.These findings suggest that Cu 2-x Se/ GO@Pt offers immense potential as a biomimetic enzyme material for determining H 2 O 2 released from living cells.This novel approach could significantly enhance our understanding of the mechanisms of ROS action in wound inflammation and pave the way for simplified monitoring of ROS dynamics in inflammatory environments.

Conclusions
In conclusion, our work has resulted in the successful synthesis of a NIR-enhanced Cu 2-x Se/GO@Pt/SPCE sensing electrode.This non-enzymatic current electrode demonstrates high selectivity, a low detection limit (0.53 mM) and wide linear range (10 mM-1500 mM) for H 2 O 2 detection.The GO nanosheets not only build a solid three-dimensional structure for the anchoring of Cu 2-x Se nanorods and the electrodeposition of Pt NPs, but also provide additional ion/ electron transport channels for electrochemical reactions.The localized surface plasma effect of Cu 2-x Se further enhances the catalytic effect of the electrode on H 2 O 2 under near-infrared illumination conditions by promoting the separation of photogenerated electron-hole pairs in the Cu 2 -x Se@Pt heterojunction.Furthermore, the Cu 2-x Se/GO@Pt/SPCE enables rapid capture of the reduction current signal of H 2 O 2 release from living cells in L929 and RAW 264.7 cell suspension, allowing for in situ real-time detection.Overall, this Cu 2-x Se/ GO@Pt/SPCE sensing electrode offers superior performance, costeffectiveness, simple preparation steps, and high reproducibility, making it a promising candidate for real-time in situ detection of biomolecules.This innovation holds the potential to transform the field of wound inflammation treatment by offering clinicians a robust method for promptly and precisely assessing ROS levels, ultimately enhancing patient outcomes.

SCHEME 1
SCHEME 1Schematic illustration of the formation of Cu2-xSe/GO@Pt/SPCE electrochemical sensors and its principle for H 2 O 2 detection.

4 (
FIGURE 4 (A) Amperometric response of Cu 2-x Se/GO@Pt/SPCE at potential of−0.4V (without laser and with 808 nm laser) with the successive addition of various amounts of H 2 O 2 ; (B) the magnified parts of (A) at low H 2 O 2 concentrations; (C) Calibration curves between electrocatalytic current of Cu 2-x Se/GO@Pt/SPCE and H 2 O 2 concentration within a range of 10−1500 mM without laser (gray line) and with 808 nm laser (red line); (D) The study of the selectivity, amperometric responses of the Cu 2-x Se/GO@Pt/SPCE to the successive dropwise additions of 0.1 mM H 2 O 2 and 1 mM interfering species (Gly, CA, AA, Urea and GSH).
FIGURE 5 (A) Demonstration of a pathway for H 2 O 2 production by human cells and a scheme for the timely capture of H 2 O 2 by sensing electrodes in response to drug stimulation; (B) i-t response of the Cu 2-x Se/GO@Pt/SPCE sensing platform to the different concentration addition of fMLP stimulation with and without living L929 cells (7*10 7 ) at an applied potential of -0.40 V in 0.01 M PBS buffer (pH=7.4);(C) i-t response of the Cu 2-x Se/GO@Pt/SPCE sensing platform to the different concentration addition of fMLP stimulation with and without living RAW 264.7 cells (8*10 7 ) at an applied potential of -0.40 V in 0.01 M PBS buffer (pH=7.4);(D) the corresponding average value of the i-t responses (n=3).